Airway hyperreactivity in diseases such as asthma involves enhanced airway smooth muscle (ASM) contraction due either to increased intracellular Ca2+ ([Ca2+]i) and/or increased Ca2+ sensitivity (force for a given [Ca2+]i). Airway inflammation is a key aspect of airways disease, and exposure to several inflammatory mediators (such as tumor necrosis factor (TNFa) and interleukin-13 (IL-13)) increase ASM contractility. Several studies showed that cytokines increase agonist-induced [Ca2+]i responses in ASM, thus linking In the current proposal, we will explore inflammation-induced changes in [Ca2+]i regulatory mechanisms that result in overall increased [Ca2+]i responses. In ASM, Ca2+ influx in response to SR depletion (store-operated Ca2+ entry; SOCE) is enhanced by cytokines. Preliminary data suggests that Na+/Ca2+ exchange (NCX)-mediated influx is enhanced by cytokines. Such enhancement of SOCE and influx-mode NCX would lead to increased [Ca2+]i levels. Reduction in [Ca2+]i is normally achieved by plasma membrane (PM) efflux mechanisms (perhaps including NCX-mediated efflux), and by SR Ca2+ reuptake via SR ATPase (SERCA). Organelles such as mitochondria can buffer Ca2+ and alter Ca2+ availability for SR refilling. Normally, these mechanisms help maintain basal [Ca2+]i at low levels, while SR Ca2+ stores are replete until agonist stimulation when [Ca2+]i rises as SR stores deplete. Preliminary studies suggest that SERCA and mitochondrial Ca2+ buffering are impaired by inflammation. Based on these contrasting preliminary findings of enhanced Ca2+ influx, but decreased sequestration or efflux, our central hypothesis is that inflammation promotes mechanisms that increase [Ca2+]i, but impairs those that decrease [Ca2+]i. This leads to an overall increase in basal [Ca2+]i as well as enhanced [Ca2+]i responses to agonist stimulation. In this regard, we propose that PM vs. intracellular mechanisms functionally interact in [Ca2+]i homeostasis under normal circumstances, and that disruption of these mechanisms and their interactions with inflammation leads to increased [Ca2+]i. Our overall approach will be to use human ASM cells or tissue strips to examine the above mechanisms with or without exposure to pro-inflammatory cytokines (TNFa, IL-13). Studies will use complementary techniques including molecular biology (siRNA; overexpression), imaging of [Ca2+]i, [Na+]i and luminal (SR) Ca2+, real-time confocal imaging of fluorescently- tagged proteins, as well as force measurements to address these aims. Our Specific Aims are: decrease [Ca2+]i Aim 1: To determine the influence of inflammatory cytokines on STIM1, STIM2 and Orai1 interactions in human ASM regulation; Aim 2: To determine the influence of inflammatory cytokines on NCX and its role in [Ca2+]i regulation in human ASM; Aim 3: To determine the influence of inflammatory cytokines on mitochondria and its role in [Ca2+]i regulation in human ASM; Aim 4: To determine the influence of inflammatory cytokines on SERCA and its role in [Ca2+]i regulation in human ASM; [Ca2+]i Aim 5: To determine the influence of inflammatory cytokines on the overall contribution of [Ca2+]i regulatory mechanisms to contractility in human ASM.